The single-gene reassortant (SGR) virus possessing a PB2 gene from an avian influenza A virus and the remaining genes from a human influenza A virus failed to produce plaques on mammalian cell monolayer culture and was restricted in replication in monkeys. Revertant viruses, which replicated and produced plaques efficiently on mammalian cells, possessed an amino acid substitution at residue 627 (glutamine in the avian PB2 gene -> lysine in the revertant) and replicated to moderate levels in monkeys. the amino acid at position 627 in every avian virus PB2 gene sequenced to date is glutamic acid and in very human virus gene it is lysine. Thus the amino acid at position 627 on the PB2 protein is an important determinant of host range of influenza virus as well as virulence of these viruses for primates. Gull-human influenza A virus reassortants derived from three different gull influenza A viruses were attenuated for squirrel monkeys or chimpanzees and, therefore, are candidate live attenuated viruses for use as vaccines in humans. Ten distinct amino acid substitutions in the human influenza A virus PB2 protein that each separately specify the temperature-sensitive phenotype were identified. Previous studies had demonstrated that influenza A viruses with ts mutations were attenuated in humans. Techniques are being developed to introduce one or more of these ts mutations into a synthetic copy of the PB2 gene and to rescue this synthetic gene into an infectious virus.